Field of the Invention
[0001] The present invention relates to an agent for suppressing elevation of blood GIP
level, an agent for suppressing elevation of blood insulin level, an agent for lowering
blood triglyceride level after meal ingestion, and an agent for suppressing elevation
of blood glucose level.
Background of the Invention
[0002] GIP (gastric inhibitory polypeptide or glucose-dependent insulinotrophic polypeptide)
is one of the gastrointestinal hormones, and is secreted from K-cells present in the
small intestine during meal ingestion. It is known that GIP functions as a suppressor
of gastric-acid secretion and suppressor of gastric motility (see Patent Literatures
1 to 3).
It is also known that GIP promotes secretion of insulin from pancreatic β-cells and
accelerates glucose uptake into adipocytes in the presence of insulin. it is therefore
considered that the functions of GIP contribute to obesity. In fact, it was reported
that obesity is suppressed when GIP functions are inhibited (see Non-patent Literature
4).
Furthermore, it was reported that GIP contributes to insulin resistance (see Non-patent
Literature 4). When insulin resistance develops, the effect of insulin on sugar uptake
decreases. As a result, hyperinsulinemia occurs, It is said that hyperinsulinemia
is a fundamental cause leading to development of various lifestyle diseases such as
obesity. Thus, it is important to prevent or reduce insulin resistance also from the
viewpoint of decreasing the risk of lifestyle diseases.
[0003] Hence, effects such as promotion of digestion, reduction of upset stomach, and prevention
or reduction of obesity and insulin resistance can be expected if GIP can be effectively
suppressed.
Previous studies show that 3-bromo-5-methyl-2-phenylpyrazolo[1,5-a]pyrimidin-7-ol
(BMPP) and pyrazolopyrimidine compounds are substances capable of inhibiting the functions
of GIP. Furthermore, guar gum and the like are known as substances capable of suppressing
postprandial secretion of GIP (see Patent Literatures 1 and 2, and Non-patent Literatures
5 to 10). However, these substances have not been found to be sufficient from the
viewpoints of safety and efficacy.
[0004] Insulin is one of the peptide hormones secreted from pancreatic β-cells in the pancreas,
and functions to decrease elevated blood glucose level and maintain it at a normal
level. The main physiological effects of insulin include promotion of uptake of sugars,
amino acids and the like, and promotion of protein synthesis in the muscle tissue;
promotion of sugar uptake and utilization thereof, promotion of lipid synthesis, suppression
of fat decomposition and burning, and promotion of protein synthesis in the adipose
tissue; and the like.
Secretion of insulin is promoted mainly by glucose. When blood sugar level (blood
glucose level) elevates owing to introduction of sugar into the body such as after
a meal, insulin is secreted so as to decrease the elevated blood sugar level. As a
result, the blood insulin level elevates. The secretion of insulin is therefore very
important for preventing diabetes mellitus by keeping blood sugar level constant.
[0005] However, it is known that continuous secretion of insulin under hyperglycemic condition
leads to insulin resistance in the skeletal muscles, liver and adipose tissue that
are target organs of insulin. When insulin resistance occurs, a larger amount of insulin
is secreted from the pancreas so as to compensate for insufficient blood sugar reduction
effect. When such an excess secretion of insulin is repeated, the pancreas gets exhausted,
and finally the ability of the pancreatic β-cells to secrete insulin declines, while
the high insulin resistance in the target organs is maintained. The above functional
deterioration of the regulatory mechanism of insulin in the body leads to constitutional
predispositions susceptible to development of lifestyle diseases such as diabetes
mellitus, and as a result, obesity, Type II diabetes mellitus (hypertension) and the
like are apt to develop (see Non-patent Literature 11).
[0006] Until recently, it has been considered that the amount of insulin secreted into the
blood varies with blood sugar level, i.e., amount of carbohydrates ingested. But,
in recent years, it was newly reported that fat uptake, as well as carbohydrate uptake,
is also correlated with the elevation of blood insulin level (see Patent Literature
3). According to Patent Literature 3, it was confirmed that ingestion of carbohydrate
together with fat induces excessive secretion of insulin beyond the level induced
by ingesting carbohydrates alone. Further, it was also ascertained that the excessive
secretion of insulin due to such simultaneous ingestion of carbohydrate and fat is
a factor highly correlated to obesity.
[0007] Meanwhile, it was reported that hyperglycemia and hyperlipidemia are independent
risk factors for cardiovascular events (Non-Patent Literatures 12 and 13). In addition,
it was reported regarding hyperglycemia that fasting hyperglycemia has a low correlation
with the probability of death caused by circulatory diseases, whereas blood sugar
level has a high correlation with the probability of death caused by circulatory diseases
in patients with hyperglycemia having a 2-hour value of 200 mg/dL or more in the glucose
tolerance test (OGTT) (Non-Patent Literature 13). It was further reported that in
the case where vascular endothelial cells are cultured in a hyperglycemic state, apoptosis
of the cells occurs with higher frequency when exposed to an intermittent hyperglycemic
condition than when exposed to a continuous hyperglycemic condition (Non-Patent Literature
14).
[0008] Further, as compared to healthy people, insulin secretion is lower in diabetes mellitus
type 1 patients and insulin secretion in the early postprandial period is lower in
diabetes mellitus type 2 patients. The postprandial blood sugar level of healthy people
is regulated by insulin, and in general, does not rise above 7.8 mmol/L (140 mg/dL)
in response to eating, and generally returns to the level before meal ingestion within
2 to 3 hours (Non-Patent Literatures 15 and 16). In contrast, in type 1 and type 2
diabetic patients with decreased insulin functions, postprandial hyperglycemia is
observed with extremely high frequency. Further, postprandial hyperglycemia is a phenomenon
which appears before clinically obvious diabetes mellitus with a progressive decrease
in insulin function and the β cell activity (a decrease in insulin secretion). It
is therefore believed that the prevention of postprandial hyperglycemia leads to the
prevention of diabetes mellitus and arteriosclerosis.
[0009] From such standpoint, administration of α-glucosidase inhibitors which delay the
digestion and absorption of carbohydrates in the small intestine, administration of
sulfonium urea formulations which promote insulin secretion, rapid-acting insulin
secretagogues and the like are performed in order to reduce postprandial hyperglycemia.
The α-glucosidase inhibitors, however, have problems in that the effect cannot be
exerted unless the inhibitors are administered before intake of carbohydrates; the
inhibitors do not affect the elevation of blood glucose level caused by intake of
glucose which is a monosaccharide; and intestinal symptoms such as diarrhea and gas
retention caused by abnormal fermentation of sugars in the colon occur. In addition,
the sulfonium urea formulations cannot exert the effect unless the formulations are
administered before intake of carbohydrates, and when the formulations are administered
excessively, excessive secretion of insulin is induced, to thereby cause hypoglycemia,
so that caution is required. Further, these synthetic medical formulations cannot
be easily obtained because prescriptions are required. In addition, the formulations
may cause various adverse effects, and for the use thereof, strict supervision and
guidance by doctors are required.
[0010] A triglyceride is a kind of neutral fat, and most neutral fats contained in blood
are triglycerides. It is known that hypertriglyceridemia and hyperlipidemia develop
with continuance of a high triglyceride level in the blood. Hyperlipidemia is considered
to be a cause of arteriosclerosis, and act as the initial trigger for inducing disorders
such as cardiac disease and cerebral vascular disease.
In general, since changes in blood triglyceride level are strongly affected by diet,
complete regulation of blood triglyceride level using only medicaments is said to
be difficult. A stronger focus has therefore been placed on the quality of ingested
dietary fat than on medical therapy. For example, lowering blood triglyceride level
by eating highly-unsaturated fatty acids, such as linoleic acid and linolenic acid,
has been recommended. But, since excessive consumption of the highly-unsaturated fatty
acids induces production of overoxidized fatty acids in vivo, the possibility of inducing
various lifestyle related diseases has been pointed out.
From the viewpoint of the above circumstances, there is a need to suppress elevation
of blood triglyceride level by a safer means which does not induce adverse effects
even if involving administration or consumption on a daily basis. Recent years, as
substances which suppress elevation of blood triglyceride level safely and effectively,
xanthane gum, propylene glycol alginate ester (see Patent literature 4), chitosan
(see Patent literature 5) and a processed starch (see Patent literature 6) have been
reported as fat absorption suppressors.
[0011] Due to their high water retaining ability, polyglutamic acids are widely used as
moisturizing agents, absorbing agents and the like in the fields of foods, medical
treatment, cosmetics and the like, and have attracted attention as highly safe biodegradable
polymers. It was reported that polyglutamic acids have an effect of promoting calcium
absorption through the small intestine and an effect of suppressing elevation of blood
pressure (for example, see Patent Literatures 7 and 8). In addition, an agent for
reducing blood sugar level using polyglutamic acids has been suggested for suppressing
elevation of blood sugar level (see Patent Literature 9). Further, it was reported
that polyglutamic acids have an effect of suppressing the absorption of neutral fat,
and can be used for the treatment, reduction and suppression of development of hypertriglyceridemia
(see Patent Literature 10).
But no pharmaceutical effects of specific salts of polyglutamic acids have been reported
so far.
Citation List
Patent Literatures
Non-Patent Literatures
[0013]
Non-Patent Literature 1: J. C. Brown et al., Canadian J. Physiol. Pharmacol., 47: 113-114, 1969
Non-Patent Literature 2: J. M. Falko et al., J. Clin. Endocrinol. Metab., 41(2): 260-265, 1975
Non-Patent Literature 3: "Gastrointestinal Tract, Function and Pathological Condition", Chugai Igakusha, p.
205-216, 1981
Non-Patent Literature 4: Nat. Med. 8(7): 738-42, 2002
Non-Patent Literature 5: Diabet Med. 13(4): 358-64,1996
Non-Patent Literature 6: Br. J. Nutr. 74(4): 539-56,1995
Non-Patent Literature 7: Reprod. Nutr. Dev. 32(1): 11-20, 1992
Non-Patent Literature 8: Br. J. Nutr. 64(1): 103-10,1990
Non-Patent Literature 9: Diabet Med. 7(6): 515-20, 1990
Non-Patent Literature 10: Br. J. Nutr. 53(3): 467-75,1985
Non-Patent Literature 11: J. Clin. Invest. 75(3): 809-17, 1985
Non-Patent Literature 12: Diabetes Care.1999; 22: 920-924
Non-Patent Literature 13: Arch. Intern. Med.2001; 161: 397-405
Non-Patent Literature 14: Am. J. Physiol. Endocrinol. Metab. 2001; 281: E924-930
Non-Patent Literature 15: Diabetes Care 2001; 24(4): 775-778
Non-Patent Literature 16: J. Clin. invest. 1988; 81(2): 442-448
Summary of the Invention
[0014] The present invention is contemplated for providing an agent for suppressing elevation
of blood GIP level which is useful for medicinal use and application in foods. Specifically,
the present invention is contemplated for providing an agent for suppressing elevation
of blood GIP level which is useful for medicinal use or non-medicinal application
in foods for decreasing the risk of development of, and preventing, reducing, alleviating
or treating, upset stomach caused by elevation of blood GIP level after meal ingestion,
for promoting digestion, or for decreasing the risk of development of, and preventing,
reducing, alleviating or treating, obesity and insulin resistance.
[0015] In addition, the present invention is contemplated for providing an agent for suppressing
elevation of blood insulin level, which can suppress elevation of blood insulin level
after meal ingestion. Further, the present invention is also contemplated for providing
an agent for suppressing elevation of blood insulin level, which is useful for preventing
or reducing obesity and diabetes mellitus caused by elevation of blood insulin level.
Specifically, the present invention is contemplated for providing an agent for suppressing
elevation of blood insulin level, which suppresses elevation of blood insulin level
after meal ingestion and is thereby useful for medicinal use or non-medicinal application
in foods for decreasing the risk of development of, and preventing, reducing, alleviating
or treating, obesity and diabetes mellitus.
[0016] Moreover, the present invention is contemplated for providing an agent for lowering
blood triglyceride level after meal ingestion, which is useful for medicinal use and
application in food. Specifically, the present invention is contemplated for providing
an agent for lowering blood triglyceride level after meal ingestion, which lowers
blood triglyceride level after meal ingestion and is thereby useful for medicinal
use or non-medicinal application in foods for decreasing the risk of development of,
and preventing, reducing, alleviating or treating, hypertriglyceridemia, hyperlipidemia
and arteriosclerosis.
[0017] in addition, the present invention is contemplated for providing an agent for suppressing
elevation of blood glucose level, which may suppress elevation of blood glucose level
after meal ingestion. Further, the present invention is also contemplated for providing
an agent for suppressing elevation of blood glucose level, which is useful for preventing
or mitigating diabetes mellitus, obesity and arteriosclerosis caused by elevation
of blood glucose level. Specifically, the present invention is contemplated for providing
an agent for suppressing elevation of blood glucose level, which suppresses elevation
of blood glucose level after meal ingestion and is thereby useful for medicinal use
or non-medicinal application in foods for decreasing the risk of development of, and
preventing, reducing, alleviating or treating, diabetes mellitus, obesity and arteriosclerosis.
[0018] The present inventors studied functions of polyglutamic acids and their salts to
suppress GIP secretion, and found that potassium polyglutamates markedly suppress
GIP secretion after meal ingestion and are particularly useful for promoting digestion,
reducing upset stomach, or preventing or reducing obesity and insulin resistance.
[0019] Further, the present inventors found that salts of polyglutamic acids have an effect
of suppressing elevation of blood GIP level after meal ingestion. The present inventors
further found that among the salts of polyglutamic acids, potassium salts have an
outstanding effect of suppressing elevation of blood insulin level.
[0020] Further, the present inventors found that salts of polyglutamic acids have an effect
of suppressing elevation of blood triglyceride level after meal ingestion. The present
inventors further found that among the salts of polyglutamic acids, potassium salts
have an effect of lowering blood triglyceride level after meal ingestion.
[0021] Further, the present inventors found that potassium polyglutamates are excellent
in effect of suppressing elevation of blood glucose level.
The present invention was completed based on the above findings.
[0022] The present invention provides the following means.
<1> Use of a potassium polyglutamate for the preparation of an agent for suppressing
elevation of blood GIP level.
<2> Use of a potassium polyglutamate for the preparation of an agent for suppressing
elevation of blood insulin level.
<3> Use of a potassium polyglutamate for the preparation of an agent for lowering
blood triglyceride level after meal ingestion.
<4> Use of a potassium polyglutamate for the preparation of an agent for suppressing
elevation of blood glucose level.
<5> The use of a potassium polyglutamate according to <1>, wherein the agent for suppressing
elevation of blood GIP level is an agent for suppressing elevation of blood GIP level
after meal ingestion.
<6> The use of a potassium polyglutamate- according to <2>, wherein the agent for
suppressing elevation of blood insulin level is an agent for suppressing elevation
of blood insulin level after meal ingestion.
<7> The use of a potassium polyglutamate according to <4>, wherein the agent for suppressing
elevation of blood glucose level is an agent for suppressing elevation of blood glucose
level after meal ingestion.
<8> A potassium polyglutamate for use in the suppression of elevation of blood GIP
level.
<9> A potassium polyglutamate- for use in the suppression of elevation of blood insulin
level.
<10> A potassium polyglutamate- for use in the lowering of blood triglyceride level
after meal ingestion.
<11> A potassium polyglutamate for use in the suppression of elevation of blood glucose
level.
<12> Non-therapeutic use of a potassium polyglutamate for the suppression of elevation
of blood GIP level.
<13> Non-therapeutic use of a potassium polyglutamate for the suppression of elevation
of blood insulin level.
<14> Non-therapeutic use of a potassium polyglutamate for the lowering of blood triglyceride
level after meal ingestion.
<15> Non-therapeutic use of a potassium polyglutamate for the suppression of elevation
of blood glucose level.
<16> A method for suppressing elevation of blood GIP level, the method including administering
a potassium polyglutamate to a subject.
<17> A method for suppressing elevation of blood insulin level, the method including
administering a potassium polyglutamate to a subject.
<18> A method for lowering blood triglyceride level after meal ingestion, the method
including administering a potassium polyglutamate to a subject.
<19> A method for suppressing elevation of blood glucose level, the method including
administering a potassium polyglutamate to a subject.
[0023] The agent for suppressing elevation of blood GIP level of the present invention can
suppress elevation of blood GIP level, specifically elevation of blood GIP level after
meal ingestion. Furthermore, the agent for suppressing elevation of blood GIP level
of the present invention can suppress elevation of blood GIP level effectively, and
is therefore useful for decreasing the risk of development of, and preventing, reducing,
alleviating or treating, upset stomach, for promoting digestion, and for decreasing
the risk of development of, and preventing, reducing, alleviating or treating, obesity
and insulin resistance.
[0024] The agent for suppressing elevation of blood insulin level of the present invention
can suppress elevation of blood insulin level, specifically elevation of blood insulin
level after meal ingestion. Furthermore, the agent for suppressing elevation of blood
insulin level of the present invention suppresses excessive elevation of blood insulin
level and therefore controls the functional mechanisms of insulin in the body after
meal ingestion within a normal range, and is thereby useful for preventing or reducing
obesity and diabetes mellitus.
[0025] The agent for lowering blood triglyceride level after meal ingestion of the present
invention can lower blood triglyceride level after meal ingestion. Furthermore, the
agent for lowering blood triglyceride level of the present invention controls blood
lipid level within a normal range, and is thereby useful for decreasing the risk of
development of, and preventing, reducing, alleviating or treating, hypertriglyceridemia
and hyperlipidermia, and for decreasing the risk of development of, and preventing,
reducing, alleviating or treating, arteriosclerosis.
[0026] The agent for suppressing elevation of blood glucose level of the present invention
can suppress elevation of glucose level, specifically elevation of blood glucose level
after meal ingestion. Furthermore, the agent for suppressing elevation of blood glucose
level of the present invention suppresses elevation of blood glucose level after meal
ingestion and therefore controls blood glucose level in the body within a normal range,
and is thereby useful for decreasing the risk of development of, and preventing, reducing,
alleviating or treating, diabetes mellitus, obesity and arteriosclerosis.
[0027] Other and further features and advantages of the invention will appear more fully
from the following description.
Detailed Description of the Invention
[0028] Hereinafter, the present invention is described in detail.
The agent for suppressing elevation of blood GIP level, the agent for suppressing
elevation of blood insulin level, the agent for lowering blood triglyceride level
after meal ingestion, and the agent for suppressing elevation of blood glucose level
of the present invention (hereinafter referred to agent of the present invention as
a superordinate concept including these agents) contain potassium salts of polyglutamic
acids (potassium polyglutamates) as active ingredients. A polyglutamic acid is a compound
in which the carboxyl group at the γ-position and the amino group at the α-position
of glutamic acids are bound to each other with a peptide bond, and a structural formula
thereof is represented by (-NH-CH(COOH)-CH
2-CH
2-CO-)n. The potassium polyglutamates used in the present invention are compounds represented
by the above structural formula except that 50% or more of the hydrogen atoms in the
carboxyl groups of the above structural formula are substituted with potassium. The
potassium polyglutamates used in the present invention are preferably compounds represented
by the above structural formula except that 60% or more, more preferably 70% or more,
more preferably 80% or more, more preferably 90% or more, more preferably 95% or more,
more preferably 99% or more of the hydrogen atoms in the carboxyl groups of the above
structural formula are substituted with potassium. It is further preferable that substantially
all of the hydrogen atoms in the carboxyl groups of the above structural formula are
substituted with potassium. Further, it is preferable that the carboxyl group positioned
at terminal end of the above structural formula is also substituted with potassium.
The potassium polyglutamates have functions of remarkably suppressing elevation of
blood GIP level, elevation of blood insulin level, elevation of blood triglyceride
level and elevation of blood glucose level, as compared to polyglutamic acids and
the other salts of polyglutamic acids. Therefore, the potassium polyglutamates can
be used as the agent of the present invention and can also be used for the preparation
of the agent of the present invention. It has not been known so far that the potassium
polyglutamates have functions of suppressing elevation of blood GIP level, suppressing
elevation of blood insulin level, lowering blood triglyceride level after meal ingestion,
suppressing elevation of blood glucose level. And also, it has not been known that
the potassium polyglutamates have an effect to prevent or reduce obesity and insulin
resistance.
[0029] The agent for suppressing elevation of blood GIP level of the present invention can
be preferably used for suppressing elevation of blood GIP level particularly after
meal ingestion. Specifically, the agent can be more preferably used for suppressing
elevation of blood GIP level after ingesting a meal containing both fat and carbohydrates,
more preferably after ingesting a meal containing a large amount of fat, more preferably
after ingesting a triacylglycerol-rich meal.
The triacylglycerol in the meal is not specifically limited. Specific examples of
the fatty component containing a large amount of triacylglycerol include butter, lard,
fish oil, corn oil, rapeseed oil, olive oil, sesame oil and the like.
The carbohydrate component in the meal is also not specifically limited. Specific
examples of the carbohydrate component include cooked rice, starch, wheat flour, sugar,
fructose, glucose, glycogen and the like.
An ingestion amounts of the fat and the carbohydrates are not specifically limited
as long as these amounts are within ranges included in a normal meal.
In the present invention, "suppression of elevation of blood GIP level" mainly refers
to suppression of elevation of blood GIP level that occurs after meal ingestion. The
suppression of the elevation of blood GIP level after meal ingestion does not necessarily
mean that the elevation of blood GIP level caused after meal ingestion is completely
suppressed, and encompasses moderating the degree of elevation of blood GIP level
as compared to the case in which the agent for suppressing elevation of blood GIP
level of the present invention is not administered. Furthermore, "function (effect)
of suppressing elevation of blood GIP level" in the present invention encompasses
both a function of suppressing secretion of GIP in which elevation of blood GIP level
is suppressed by suppressing GIP secretion from the digestive tract, and a function
of lowering GIP in which elevation of blood GIP level is suppressed by lowering the
blood GIP level.
[0030] In the present invention, "suppressing elevation of blood insulin level" means suppression
of elevation of blood insulin level caused by ingesting foods containing primarily
fat and carbohydrate.
The suppression of the elevation of blood insulin level after meal ingestion does
not necessarily mean that the elevation of blood insulin level caused after meal ingestion
is completely suppressed, and encompasses moderating the degree of elevation of blood
insulin level as compared to the case in which the agent for suppressing elevation
of blood insulin level of the present invention is not administered.
Furthermore, in the present invention, "function (effect) of suppressing elevation
of blood insulin level" encompasses both a function of suppressing secretion of insulin
in which elevation of blood insulin level is suppressed by suppressing secretion of
insulin from the pancreas, and a function of lowering insulin in which elevation of
blood insulin level is suppressed by lowering the blood insulin level.
[0031] The fat described above is not specifically limited as long as it is fatty component
included in a normal meal and capable of promoting insulin secretion. Specific examples
include butter, lard, fish oil, com oil, rapeseed oil, olive oil, sesame oil and the
like.
The carbohydrates described above are not specifically limited as long as it is a
carbohydrate component that is included in a normal meal and promote secretion of
insulin. Specific examples include cooked rice, starch, wheat flour, sugar, fructose,
glucose, glycogen and the like.
It is also known that, when the above-mentioned carbohydrates are ingested with fat,
the elevation of blood insulin level is enhanced as compared to the case in which
carbohydrates are ingested solely. The agent for suppressing elevation of blood insulin
level of the present invention can effectively suppress rapid elevation of blood insulin
level caused after ingesting both the fat and carbohydrates described above. Thus,
for example even when fat and carbohydrates are ingested in an amount of not less
than 5 g/60 kg body weight and not less than 10 g/60 kg body weight respectively in
a meal, the agent for suppressing elevation of blood insulin level of the present
invention can suppress (approach) elevation of blood insulin level to a normal degree
of elevation.
[0032] As shown in Examples described below, the agent for lowering blood triglyceride level
after meal ingestion of the present invention can lower blood triglyceride level after
meal ingestion (after ingesting foods, beverages and the like, which contain general
carbohydrates, fat, proteins etc).
[0033] In the present invention, "suppressing elevation of blood glucose level" mainly means
suppression of elevation of blood glucose level after meal ingestion. The suppression
of the elevation of blood glucose level after meal ingestion does not necessarily
mean that the elevation of bbod glucose level caused after meal ingestion is completely
suppressed, and encompasses moderating the degree of elevation of blood glucose level
as compared to the case in which the agent for suppressing elevation of blood glucose
level of the present invention is not administered.
[0034] The term "reduction" in the present specification encompasses changing for the better,
preventing deterioration, delaying progression, reversal of progression and preventing
progression of diseases, symptoms or conditions.
[0035] The term "prevention" in the present specification means that the developments of
diseases or symptoms in individuals are prevented or delayed, or the risk of development
of diseases or symptoms in individuals are lawered.
[0036] The molecular weight of the potassium polyglutamates used in the present invention
is preferably a weight-average molecular weight of about 500 or more, more preferably
of 1,000 or more, more preferably of 2,000 or more, more preferably of 3,000 or more,
more preferably of 5,000 or more. Although, the upper limit of the weight-average
molecular weight of the potassium polyglutamates used in the present invention is
preferably about 5,000,000, it is preferable that the preparation has a comparatively
lower viscosity from the viewpoints of preparation, and of feeling of the throat,
slimy feeling, easiness of swallowing and the like when the agent for suppressing
elevation of blood insulin level of the present invention is used in the form of an
oral liquid preparation. Thus, the weight-average molecular weights of the potassium
polyglutamates used in the present invention is more preferably 1,000,000 or less,
more preferably 500,000 or less.
[0037] Although the effect of suppressing elevation of blood GIP level by the potassium
polyglutamates is observed in overall potassium polyglutamates regardless of their
molecular weight, the potassium polyglutamates having larger molecular weights to
some extent are more excellent in an effect of suppressing elevation of blood GIP
level.
Thus, the weight-average molecular weight of the potassium polyglutamates used in
the present invention is preferably from 500 to 5,000,000, more preferably from 1,000
to 1,000,000, more preferably from 2,000 to 500,000, more preferably from 3,000 to
500,000, more preferably from 4,000 to 400,000, more preferably from 5,000 to 300,000,
more preferably from 6,000 to 240,000, in order to more effectively suppress elevation
of blood GIP level. The weight-average molecular weight can be measured with, for
example, high performance liquid chromatography using a gel filtration column.
[0038] Although the effect of suppressing elevation of blood insulin level by the potassium
polyglutamates is observed in overall potassium polyglutamates regardless of their
molecular weight, the potassium polyglutamates having too large molecular weight tend
to show a less effect of suppressing elevation of blood insulin level.
The potassium polyglutamates used in the agent for suppressing elevation of blood
insulin level of the present invention show an extremely higher effect of suppressing
elevation of blood insulin level as compared to polyglutamic acids and metal salts
thereof other than potassium salts which have a weight-average molecular weight similar
to the potassium polyglutamates. The weight-average molecular weight of the potassium
polyglutamates used in the agent for suppressing elevation of blood insulin level
of the present invention is preferably from 500 to 1,000,000. In order to more effectively
suppress elevation of blood insulin level, the weight-average molecular weight is
more preferably from 1,000 to 800,000, more preferably from 2,000 to 400,000, more
preferably from 3000 to 200,000, more preferably from 4,000 to 150,000, more preferably
from 5,000 to 50,000, more preferably from 5,000 to 20,000, more preferably from 6,000
to 12,000.
[0039] Although the effect of lowering blood triglyceride level after meal ingestion by
the potassium polyglutamates is observed in overall potassium polyglutamate regardless
of their molecular weight, the potassium polyglutamates having a lower molecular weight
tend to show a higher effect.
The weight-average molecular weight of the potassium polyglutamates used in the agent
for lowering blood triglyceride level after meal ingestion in the present invention
is preferably from 500 to 5,000,000, more preferably from 1,000 to 1,000,000, more
preferably from 2,000 to 500,000, more preferably from 3,000 to 500,000, more preferably
from 4,000 to 400,000, more preferably from 5,000 to 300,000, more preferably from
6,000 to 240,000.
[0040] The effect of suppressing elevation of blood glucose level by the potassium polyglutamates
is observed in overall potassium polyglutamates regardless of their molecular weight.
The weight-average molecular weight of the potassium polyglutamates used in the agent
for suppressing elevation of blood glucose level of the present invention is preferably
from 500 to 5,000,000, more preferably from 1,000 to 1,000,000, more preferably from
2,000 to 500,000, more preferably from 3,000 to 500,000, more preferably from 6,000
to 500,000, more preferably from 8,000 to 400,000, more preferably from 10,000 to
300,000, more preferably from 12,000 to 240,000.
[0041] The potassium polyglutamates used in the present invention can be obtained by neutralizing
polyglutamic acids or salts thereof produced by chemical synthesis or microorganisms,
or commercially available polyglutamic acids or salts thereof, using an aqueous solution
of potassium hydroxide as described in Examples described below. Further, the potassium
polyglutamates can be also produced by microorganisms cultured in a mixed medium containing
potassium. The optical activity of glutamic acids that constitute the potassium polyglutamates
may be dextro-rotatory or levo-rotatory, and also may be a mixture thereof. A natural
polyglutamic acid is a polymer in which glutamic acids are bound to each other at
their γ-position, and examples of wild type microorganisms that produce polyglutamic
acids may include a part of bacteria belonging to genus
Bacillus including
Bacillus subtilis var.
natto and related species thereof (
Bacillus subtilis var.
chungkookjang,
Bacillus licheniformis, Bacillus megaterium, Bacillus anthracis, Bacillus halodurans),
Natrialba aegyptiaca, Hydra and the like [
Ashiuchi, M., et al.: Appl. Microbiol. Biotechnol., 59, pp. 9-14 (2002)]. As examples of the production of polyglutamic acids using a gene recombination
technique, it has been known that a recombinant
Bacillus subtilis ISW1214 strain, which is constructed by gene transfer with a plasmid, produces polyglutamic
acids in an amount of about 9 g/L/5 days [
Ashiuchi, M., et al.: Biosci. Biotechnol. Biochem., 70, pp. 1794-1797 (2006)], and a recombinant
E. coli, which is constructed by gene transfer with a plasmid, produces polyglutamic
acids in an amount of about 4 g/L/1.5 days [
Jiang, H., et al.: Biotechnol. Lett., 28, pp. 1241-1246 (2006)]. Furthermore, polyglutamic acids are commercially produced as food additives, materials
for cosmetics and thickening agents, and the like, and it is also possible to purchase
polyglutamic acids supplied by domestic or foreign manufacturers of polyglutamic acids
(for example, domestic manufacturers: Nippon Poly-Glu Co., Ltd., Ichimaru Pharcos
Co., Ltd., Meiji Food Materia Co., Ltd.. and the like, foreign manufacturers: BioLeaders
Corporation and the like).
[0042] The potassium polyglutamates used in the present invention can suppress GIP secretion,
insulin secretion, elevation of blood triglyceride level and elevation of blood glucose
level, after ingesting carbohydrates, fat and/or proteins. Moreover, the suppression
effects are remarkably higher than those of polyglutamic acids and other salts of
polyglutamic acids.
[0043] The agent of the present invention may be the above described potassium polyglutamates
themselves. In addition, the agent of the present invention may contain adequate liquid
or solid excipients or expanders, such as titanium oxide, calcium carbonate, distilled
water, lactose and starch, in addition to potassium polyglutamates. Although the content
of the potassium polyglutamates in the agent of the present invention is not specifically
limited, the content is preferably from 0.01 to 100% by mass, more preferably from
0,1 to 95% by mass, further preferably from 1 to 90% by mass, particularly preferably
from 5 to 85% by mass.
[0044] When the agent of the present invention is used as foods, medicaments or the like,
the potassium polyglutamates can be administered alone to humans and animals by gastrointestinal
administration, intraperitoneal administration, intravascular administration, intradermal
administration, subcutaneous administration or the like, or can be ingested as a form
of various foods, medicinal products, pet foods or the like, into which the potassium
polyglutamates are blended as active ingredients.
As the foods, in addition to general foods, it is possible to apply to foods such
as cosmetic foods, foods for diseased persons and foods for specified health use,
which have the concepts of decreasing the risk of development of, and preventing,
reducing, alleviating or treating, upset stomach, of enhancing digestion, and of decreasing
the risk of development of, and preventing, reducing, alleviating or treating, obesity
and insulin resistance, in the case of the agent for suppressing elevation of blood
GIP level; the concepts of suppressing elevation of an insulin level in the blood,
and of decreasing the risk of development of, and preventing, reducing, alleviating
or treating, obesity and diabetes mellitus, in the case of the agent for suppressing
elevation of blood insulin level; the concepts of lowering blood triglyceride level
after meal ingestion, and of decreasing the risk of development of, and preventing,
reducing, alleviating or treating, hypertriglyceridemia, hyperlipidemia and arteriosclerosis,
in the case of the agent for lowering blood triglyceride level after meal ingestion;
and the concepts of suppressing elevation of blood glucose level, and of decreasing
the risk of development of, and preventing, reducing, alleviating or treating, obesity
and diabetes mellitus, in the case of the agent for suppressing elevation of blood
glucose level, respectively, and indicate those effects as necessary. When used as
a medicinal product, the agent can be formed into an oral solid formulation such as
a tablet and a granule agent, or an oral liquid formulation such as an oral liquid
agent and a syrup agent.
[0045] When preparing the oral solid formulation as the agent of the present invention,
a tablet, a coated tablet, a granular agent, a powder agent, a capsule agent or the
like can be produced by a conventional method after blending an excipient, and if
needed, a binder, a disintegrating agent, a lubricating agent, a coloring agent, a
taste masking agent, a flavoring agent and the like to a potassium polyglutamates.
Alternatively, when preparing the oral liquid formulation, an oral liquid agent, a
syrup agent, an elixir agent or the like can be prepared by a conventional method
by blending in a taste masking agent, a buffering agent, a stabilizer, a taste masking
agent and the like.
[0046] Although the content of the potassium polyglutamates in each of the above-mentioned
foods and formulations is not specifically limited, the content is preferably from
0.01 to 100% by mass, more preferably from 0.03 to 90% by mass, more preferably from
0.1 to 80% by mass, more preferably from 0.3 to 70% by mass, more preferably from
1 to 60% by mass.
[0047] The effective administration (ingestion) amount (dosage) of the potassium polyglutamates
in each of the above-mentioned foods and formulations is preferably from 0.001 to
1.0 g/kg body weight per day, more preferably from 0.003 to 0.5 g/kg body weight per
day, more preferably from 0.01 to 0.2 g/kg body weight per day.
[0048] The agent of the present invention is preferably administered or ingested during
or before or after meal ingestion, and can be administered or ingested more preferably
during the period from 30 minutes before starting meal ingestion to 30 minutes after
finishing the meal ingestion, and more preferably during the period from 10 minutes
before starting meal ingestion to 10 minutes after finishing the meal ingestion.
[0049] The term "after meal ingestion" in the present specification indicates a period from
just after finishing meal ingestion to a time when most of carbohydrates in the meal
are absorbed from the intestine. More specifically, it indicates a period from just
after finishing meal ingestion (0 min) to 6 hours after finishing the meal ingestion,
preferably from just after finishing meal ingestion (0 min) to 5 hours after finishing
the meal ingestion, more preferably from just after finishing meal ingestion (0 min)
to 4 hours after finishing the meal ingestion, and further preferably from just after
finishing meal ingestion (0 min) to 3 hours after finishing the meal ingestion (
Diabete Care, 2001, 24 (4): 775-778).
[0050] The present invention also provides, as one aspect thereof, use of potassium polyglutamates
for the preparation of an agent for suppressing elevation of blood GIP level, of an
agent for suppressing elevation of blood insulin level, of an agent for lowering blood
triglyceride level, after meal ingestion, and of an agent for suppressing elevation
of blood glucose level.
Further, the present invention also provides, as another aspect thereof, potassium
polyglutamates for use in the suppression of elevation of blood GIP level, in the
suppression of elevation of blood insulin level, in the lowering of blood triglyceride
level after meal ingestion, and in the suppression of elevation of blood glucose level.
Further, the present invention also provides, as further another aspect thereof, non-therapeutic
use of potassium polyglutamates for the suppression of elevation of blood GIP level,
for the suppression of elevation of blood insulin level, for the lowering of blood
triglyceride level after meal ingestion, and for the suppression of blood glucose
level. The "non-therapeutic use" in the present invention may be use in human or non-human
animals, or specimens derived therefrom. In addition, the "non-therapeutic" is a concept
that does not encompass medical practices, namely, does not encompass treatments for
human bodies and non-human animals for the purpose of treatment which are conducted
by doctors or medical workers.
Further, the present invention also provides, as further another aspect thereof, a
method for suppressing elevation of blood GIP level, a method for suppressing elevation
of blood insulin level, a method for lowering blood triglyceride level after meal
ingestion, and a method for suppressing elevation of blood glucose level, these methods
including administering potassium polyglutamates to a subject.
[0051] Although subjects of administration or ingestion of the agent of the present invention
are not specifically limited as long as the subjects are in need thereof, obese persons
and persons with metabolic syndrome, and potential patients thereof are preferable
as the subjects of the administration or ingestion. In Japan, since a recommended
standard BMI is 22, subjects having BMI = 22 or more are preferable and subjects having
BMI = 25 or more are more preferable. In Europe and the United States, since BMI of
25 or more is considered to be overweight in a criterion for obesity, subjects having
BMI = 25 or more are preferable, and subjects having BMI = 30 or more are more preferable.
In a diagnostic criterion for metabolic syndrome in Japanese, a man having a waist
measuring of 85 cm or more or a woman having a waist measuring of 90 cm or more, who
meets at least one of the following three criteria falls within a potential patient,
or the man or woman who meets at least two of the following criteria falls within
metabolic syndrome: (1) blood triglyceride level is 150 mg/dl or more or HDL cholesterol
level is less than 40 mg/dL, (2) being hyperglycemia (having a fasting blood sugar
level of 110 mg/dL or more), and (3) being hypertension (130/85 mHg or more). Thus,
these persons are preferable as the subjects of the administration or ingestion. In
the United States, a person who meets three or more of the following criteria: an
abdominal girth (102 cm or more for men and 88 cm or more for women), a high neutral
fat, a low HDL, hypertension, and a high fasting-blood-sugar, falls within metabolic
syndrome. Thus, these persons including persons who meet two or more of the above
criteria and fall within a potential patient are preferable as the subjects of the
administration or ingestion.
In the case of the agent for suppressing elevation of blood GIP level, it is preferable
that subjects of administration or ingestion are persons having a fasting blood glucose
level of 100 mg/dL or more, or having fasting blood triglyceride level of 100 mg/dL
or more, and / or having fasting blood GIP level of 10 pg/mL or more.
In the case of the agent for suppressing elevation of blood insulin level, it is preferable
that subjects of administration or ingestion are persons having fasting blood glucose
level of 100 mg/dL or more, or having fasting blood triglyceride level of 100 mg/dL
or more.
In the case of the agent for lowering blood triglyceride level after meal ingestion,
it is preferable that subjects of administration or ingestion are persons having fasting
blood triglyceride level of 100 mg/dL or more.
In the case of the agent for suppressing elevation of blood glucose level, it is preferable
that subjects of administration or ingestion are persons having fasting blood glucose
level of 100 mg/dL or more, or having fasting blood triglyceride level of 100 mg/dL
or more. Further, it is preferable that subjects of administration or ingestion are
persons having blood glucose level of 140 mg/dL or more at 2 hours after finishing
meal ingestion.
Examples
[0052] Hereinafter, the present invention will be described more in detail with reference
to Examples, but the present invention is not limited thereto.
[analysis procedures]
Method for quantifying potassium polyglutamate and method for determining weight-average
molecular weight of potassium polyglutamate -1:
[0053] Quantification and weight-average molecular weight determination of potassium polyglutamates
were performed by the means of gel permeation with D-6000 HPLC system (manufactured
by Hitachi High-Technologies Corporation). The analysis conditions were that TSKGel
G4000PWXL and TSKGel G6000PWXL gel permeation columns (trade names, manufactured by
Tosoh Corporation) were used as an analytical column, 0.1 M sodium sulfate was used
as an eluent, the flow rate was 1.0 mL/min, the column temperature was 50°C and the
wavelength of UV for detection was 210 nm. Concentrations were calibrated by preparing
a calibration curve using polyglutamic acids having a molecular weight of 880 k (manufactured
by Meiji Food Materia Co., Ltd.). Weight-average molecular weight was measured using
polyglutamic acids having various different molecular weights (those manufactured
by Wako Pure Chemical Industries, Ltd. (trade name: 162-21411 and 162-21401), SIGMA-ALDRICH
(trade name: P-4886 and P-4761) and Meiji Food Materia Co., Ltd. (molecular weight
of 880 k)) as standard materials, the weight-average molecular weights of these polyglutamic
acids being obtained in advance by using pulfulan (trade name: Shodex STANDARD P-82,
manufactured by Showa Denko K.K.).
Method for determining weight-average molecular weight of potassium polyglutamate
-2:
[0054] Weight-average molecular weight determination of potassium polyglutamates with low
molecular weights having molecular weights of around 10 k was performed by the means
of gel permeation with D-6000 HPLC system (manufactured by Hitachi High-Technologies
Corporation). The analysis conditions were that TSKGel G3000PWXL permeation columns
(trade names, manufactured by Tosoh Corporation) were used as an analytical column,
0.1 M sodium sulfate was used as an eluent, the flow rate was 0.8 mL/min, the column
temperature was 50°C and the wavelength of UV for detection was 210 nm. Weight-average
molecular weight was measured using polyglutamic acids (manufactured by Meiji Food
Materia Co., Ltd., molecular weight of 9 k) and poly-hydroxyproline (manufactured
by SIGMA-ALDRICH, molecular weight of 4 k) as standard materials, the weight-average
molecular weights thereof being obtained in advance by using pullulan (trade name:
Shodex STANDARD P-82, manufactured by Showa Denko K.K.).
Metal-analysis method for salts of polyglutamic acids:
[0055] The metal analysis for salts of polyglutamic acids was performed by the means of
ion chromatography using D-7000 HPLC system (manufactured by Hitachi High-Technologies
Corporation). As the analysis conditions, Shodex IC YK-G as a guard column and Shodex
IC YK-421 as an analysis column (both are trade names, manufactured by Showa Denko
K.K.) were used, 1.5 mM citric acid aqueous solution was used as an eluent, and detection
was carried out at a flow rate of 1.0 mL/min at a column temperature of 40°C using
an electric conductivity detector. A sodium standard solution (1000 ppm) and a potassium
standard solution (1000 ppm), manufactured by KANTO CHEMICAL CO.,INC., were used as
standard samples, and the calibration curves of these standard substances were prepared
in a range from 10 to 100 mg/L. The metal analysis for salts of polyglutamic acids
was carried out based on these calibration curves.
[Preparation Example 1] Preparation of potassium polyglutamate having weight-average
molecular weight of 12,000
[0056] Using sodium polyglutamates having weight-average molecular weight of 9,000 (manufactured
by Meiji Food Materia Co., Ltd.) as starting materials, 500 mL of 10(w/w)% aqueous
solution thereof was prepared. Then, the pH of this solution was adjusted to 2 or
less with hydrochloric acid under ice-cooling. After that, the generated acid precipitate
was collected by centrifugation (trade name: himac CR21 Glll, manufactured by Hitachi
Koki Co., Ltd.) at 8,000 rpm for 5 minutes. The obtained precipitate was washed with
an equal amount of distilled water, and subjected to centrifugation once more. This
washing operation was repeated twice, and the obtained precipitate was then suspended
in 300 mL of distilled water, followed by neutralization with an aqueous solution
of potassium hydroxide to be pH 7 or more. The above-mentioned acid treatment and
neutralization treatment with potassium hydroxide were carried out once more, and
to the resulting neutralized sample, 2.5 times higher amount of ethanol was added,
and the sample was left to stand overnight under ice-cooling. The precipitate generated
by the addition of the ethanol was collected by centrifugation (same as above) at
14,000 rpm for 5 minutes, and the collected sample was subjected to drying under reduced
pressure to obtain 27.4 g of a solid sample. The weight-average molecular weight of
this sample was calculated as 12,000 by the analysis procedures described above. In
addition, potassium was detected in an amount equivalent to the amount of the carboxyl
groups of polyglutamic acids in this sample, and the amount of sodium was under a
detection limit. It was therefore confirmed that substantially all hydrogen atoms
of the carboxyl groups were substituted with potassium.
[Preparation Example 2] Preparation of potassium polyglutamate having weight-average
molecular weight of 240,000
[0057] Using sodium polyglutamates having weight-average molecular weight of 350,000 (manufactured
by Meiji Food Materia Co., Ltd.) as starting materials, 1 L of 10(w/w)% aqueous solution
thereof was prepared. Then, the pH of this solution was adjusted to 1 or less with
hydrochloric acid under ice-cooling. After that, the generated acid precipitate was
collected by centrifugation (trade name: himac CR21 GIII, manufactured by Hitachi
Koki Co., Ltd.) at 8,000 rpm for 5 minutes. The obtained precipitate was washed with
an equal amount of distilled water, and subjected to centrifugation once more. This
washing operation was repeated twice, and the obtained precipitate was then suspended
in 800 mL of distilled water, followed by neutralization with an aqueous solution
of potassium hydroxide to be pH 7 or more. The above-mentioned acid treatment and
neutralization treatment with potassium hydroxide were carried out once more, and
the precipitate generated by the addition of ethanol was collected by centrifugation
(same as above) at 14,000 rpm for 5 minutes, and the collected sample was subjected
to drying under reduced pressure to obtain 36.2 g of a solid sample. The weight-average
molecular weight of this sample was calculated as 240,000 by the analysis procedures
described above. In addition, potassium was detected in an amount equivalent to the
amount of the carboxyl groups of polyglutamic acids in this sample, and the amount
of sodium was under a detection limit. It was therefore confirmed that substantially
all hydrogen atoms of the carboxyl groups were substituted with potassium.
[Preparation Example 3] Preparation of potassium polyglutamate having weight-average
molecular weight of 6,000
[0058] Using sodium polyglutamates having weight-average molecular weight of 9,000 (manufactured
by Meiji Food Materia Co., Ltd.) as starting materials, 125 mL of 20(w/w)% aqueous
solution thereof was prepared. Then, the pH of this solution was adjusted to 1 or
less with hydrochloric acid. After that, this PGA solution was incubated at 95 °C
for 12 hours and generated acid precipitate was collected by centrifugation (trade
name: himac CR21 GIII, manufactured by Hitachi Koki Co., Ltd.) at 8,000 rpm for 5
minutes. The obtained precipitate was washed with an equal amount of distilled water,
and subjected to centrifugation once more, This washing operation was repeated twice,
and the obtained precipitate was then suspended in 300 mL of distilled water, followed
by neutralization with an aqueous solution of potassium hydroxide to be pH 7 or more.
The above-mentioned acid treatment and neutralization treatment by potassium hydroxide
were carried out once more, and to the resulting neutralized sample, 2.5 times higher
amount of ethanol was added, and the sample was left to stand overnight under ice-cooling.
The precipitate generated by the addition of the ethanol was collected by centrifugation
(same as above) at 14,000 rpm for 5 minutes, and the collected sample was subjected
to drying under reduced pressure to obtain 16.5 g of a solid sample. The weight-average
molecular weight of this sample was calculated as 6,000 by the analysis procedures
described above. In addition, potassium was detected in an amount equivalent to the
amount of the carboxyl groups of polyglutamic acids in this sample, and the amount
of sodium was under a detection limit. It was therefore confirmed that substantially
all hydrogen atoms of the carboxyl groups were substituted with potassium.
[Test Example 1] Pharmacological function of potassium polyglutamate
[0059] Samples for oral administration were prepared using potassium polyglutamates with
weight-average molecular weights of 12,000 and 240,000 (prepared in Preparation Examples
1 and 2, respectively) and sodium polyglutamates with weight-average molecular weights
of 9,000 and 350,000 (manufactured by Meiji Food Materia Co., Ltd.) as described below.
In addition, oral administration was carried out using ten 8-week-old male mice (C57BL/6J
Jcl: manufactured by CLEA Japan, Inc.) per each group as described below, followed
by collecting blood, and blood GIP levels, blood insulin levels, blood triglyceride
levels and blood glucose levels were measured by procedures described below.
[Test Example 2] Pharmacological function of potassium polyglutamate with low molecular
weight
[0060] Samples for oral administration were prepared using potassium polyglutamates with
weight-average molecular weight of 6,000 (prepared in Preparation Example 3), potassium
polyglutamates with weight-average molecular weight of 12,000 (prepared in Preparation
Example 1), and sodium polyglutamates with weight-average molecular weight of 9,000
(manufactured by Meiji Food Materia Co., Ltd.) as described below. In addition, oral
administration was carried out using seven 8-week-old male mice (C57BL/6J Jcl: manufactured
by CLEA Japan, Inc.) per each group as described below, followed by collecting blood,
and blood GIP levels, blood insulin levels, blood triglyceride levels were measured
by procedures described below.
Preparation of samples for oral administration:
[0061] An emulsion liquid was prepared by emulsifying glucose (manufactured by Kanto Kagaku)
and triolein (Glyceryl trioleate: manufactured by Sigma) using lecithin (made from
eggs, manufactured by Wako Pure Chemical Industries) and albumin (derived from bovine
serum, manufactured by Sigma). A sample for oral administration was prepared by adding
the salts of polyglutamic acids to this emulsion liquid so that the final concentrations
be 5 (w/w)% of the salts of polyglutamic acids, 5 (w/w)% of glucose, 5 (w/w)% of triolein,
and emulsifier (0.2 (w/w)% of lecithin and 1.0 (w/w)% of albumin). A sample in which
water had been added instead of the salts of polyglutamic acids was prepared as a
control sample.
Oral administration and collection of blood:
[0062] The initial blood sampling was conducted from the orbital vein of a mouse that had
been food-deprived overnight, using a heparin-treated hematocrit capillary (manufactured
by VITREX) under ether anesthesia. Thereafter, the oral administration sample was
administered orally by using a feeding needle, and after 10 minutes, 30 minutes, 1
hour and 2 hours, the blood was collected from the orbital vein under ether anesthesia.
The amount of oral administration to the mouse is shown in the following Table 1.
[0063]
Table 1 Amount of oral administration to mouse
|
Glucose (mg/1g body weight) |
Triolein (mg/1g body weight) |
Salt of polyglutamic acid (mg/1g body weight) |
Control group |
2 |
2 |
- |
Group administered salt of polyglutamic acid |
2 |
2 |
2 |
Measurement and evaluation of GIP level in blood
[0064] The blood collected by the heparin-treated hematocrit capillary was stored under
ice-cooling until blood plasma separation, and centrifuged at 11,000 rpm for 5 minutes
to give blood plasma. Blood GIP level in the obtained plasma was measured by using
a Rat/Mouse GIP (Total) ELISA kit (manufactured by Linco Research/Millipore co., ELISA
method).
The GIP levels in bloods up to 2 hours after the oral administration of the sample
were measured. As a result, it was found that the blood GIP level became the maximum
at 10 minutes after the administration. Thus, the difference (Δ value) between the
maximum value (at 10 minutes after the administration) and the initial value (at the
time of the initial blood collection) of the blood GIP level was defined as the maximum
GIP-level elevation, and Table 2 (results of Test Example 1) and Table 3 (results
of Test Example 2) show the values when the value obtained from the control group
defined as 100.
[0065] The statistically-significant difference between the groups was also studied based
on the obtained values of the maximum GIP-level elevation, and is shown in Table 2
and Table 3. When significance (p<0.05) was recognized by an analysis of variance,
verifications were performed between the salts of polyglutamic acids-administered
groups and the control group, and between the sodium salts-administered groups and
similar molecular weight of the potassium salts-administered groups using a multiple
comparison test (Bonferroni/Dunn method). From the obtained result, significance was
judged with considering p<0.05 as a significant difference.
[0066]
Table 2 Relative values of maximum GIP-level elevation (10-minute value - initial
value)
|
Maximum GIP-level elevation Average ± S.E. (pg/ml) |
Significant difference from control group |
Significant difference between sodium polyglutamate-administered group and potassium
polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
102.9 ± 5.3 |
N.S. |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
60.7 ± 4.9 |
P<0.05 |
Sodium polyglutamate (weight-average molecular weight: 350,000)-administered group |
55.6 ± 3.1 |
P<0.05 |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 240,000)-administered group |
40.1 ± 2.8 |
P<0.05 |
*) S.E. : Standard Error
*) N.S.: Not Significant |
[0067] From the results of Table 2, it was found that the elevation of blood GIP level was
not suppressed in the group to which the sodium polyglutamate of relatively low molecular
weight having a weight-average molecular weight of 9,000 was administered. In contrast,
the results showed that by increasing the weight-average molecular weight of the sodium
polyglutamates to approximately 4-fold, namely, 350,000, the elevation of blood GIP
level could be suppressed by about 45% (to about 55% with respect to the control)
as compared to the control group. Further, the results also showed that by using the
potassium polyglutamates having about one sixth weight-average molecular weight (weight-average
molecular weight of 12,000), the elevation of blood GIP level could be suppressed
by about 40% (to about 60% with respect to the control), which was almost equal suppression
level to the above. Further, it was found that by increasing the weight-average molecular
weight to 240,000, the elevation of blood GIP level could be suppressed by about 60%
(to about 40% with respect to the control).
[0068]
Table 3 Relative values of maximum GIP-level elevation (10-minute value - initial
value)
|
Maximum GIP-level elevation Average ± S.E. (pg/ml) |
Significant difference from control group |
Significant difference between sodium polyglutamate (weight-average molecular weight:
9,000)-administered group and potassium polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
102.8 ± 8.0 |
N.S. |
- |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
50.8 ± 4.1 |
P<0.05 |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 6,000)-administered group |
58.2 ± 4.8 |
P<0.05 |
P<0.05 |
*) S.E. : Standard Error
*) N.S. : Not Significant |
[0069] From the results of Table 2, it was found that the elevation of blood GIP level
was not suppressed in the group to which the sodium polyglutamates of relatively low
molecular weight having a weight-average molecular weight of 9,000 was administered.
In contrast, the elevation of blood GIP level could be suppressed by about 50% when
using the potassium polyglutamates having a weight-average molecular weight of 12,000,
and the results of the above-mentioned Test Example 1 were reproduced.
In addition, the results showed that the potassium polyglutamates with lower molecular
weight having a weight-average molecular weight of 6,000, also had the function of
decreasing GIP to the level similar to that obtained by using potassium polyglutamates
with a weight-average molecular weight of 12,000.
[0070] From the results of Test Example 2, it was found that the salts of polyglutamic acids
had an effect of suppressing elevation of blood GIP level, and among these, potassium
polyglutamates exerted a remarkably excellent effect of suppressing elevation of blood
GIP level in the wide range of molecular weights.
[0071] As mentioned above, it is known that GIP has the effects of suppressing secretion
of gastric acid, suppressing gastric motility, promoting uptake of glucose into adipocyte
under the presence of insulin, and inducing insulin resistance. Thus, the above-mentioned
potassium polyglutamates can be preferably used for promoting digestion, for preventing
or reducing upset stomach, and for preventing or reducing obesity and insulin resistance
by effectively suppressing elevation of blood GIP level.
Measurement and evaluation of insulin level in blood:
[0072] The blood collected by the heparin-treated hematocrit capillary was stored under
ice-cooling until blood plasma separation, and centrifuged at 11,000 rpm for 5 minutes
to give blood plasma. Blood insulin level in the obtained plasma was measured using
an Insulin measurement kit (manufactured by Morinaga Institute of Biological Science,
Inc., ELISA Method).
The insulin levels in bloods up to 2 hours after the oral administration of the sample
were measured. As a result, it was found that the blood insulin level became the maximum
at 10 minutes after the administration. Thus, the difference (A value) between the
maximum value (at 10 minutes after the administration) and the initial value (at the
time of the initial blood sampling) of the blood insulin level was defined as the
maximum insulin-level elevation, and Table 4 (results of Test Example 1) and Table
5 (results of Test Example 2) show the values when the value obtained from the control
group defined as 100.
[0073] Further, the statistically-significant difference between the groups was also considered
based on the obtained values of the maximum insulin-level elevation, and is shown
in Table 4 and Table 5. When significance (p<0.05) was recognized by an analysis of
variance, verifications were performed between the salts of polyglutamic acids-administered
groups and the control group, and between the sodium salts-administered groups and
similar molecular weight of the potassium salts-administered groups using a multiple
comparison test (Bonferroni/Dunn method). From the obtained result, significance was
judged with considering p<0.05 as a significant difference.
[0074]
Table 4 Relative values of maximum insulin-level elevation in mouse (10-minute value
- initial value)
|
Maximum insulin-level elevation Average ± S.E. (pg/ml) |
Significant difference from control group |
Significant difference between sodium polyglutamate-administered group and Potassium
polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
103.3 ± 12.7 |
N.S. |
P<0,05 |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
45.2 ± 5.2 |
P<0.05 |
Sodium polyglutamate (weight-average molecular weight: 350,000)-administered group |
86.2 ± 8.2 |
N.S. |
N.S. |
Potassium polyglutamate (weight-average molecular weight of 240,000)-administered
group |
78.3 ± 6.0 |
P<0.05 |
*) S.E. : Standard Error
*) N.S. : Not Significant |
[0075] From the results shown in Table 4, it was found that the sodium polyglutamates did
not have a significant function of suppressing elevation of blood insulin level. In
addition, it was also found that although the sodium polyglutamates-administered groups
were not significantly different from the control group, those with a larger molecular
weight increased an effect of suppressing elevation of blood insulin level. In contrast,
the potassium polyglutamates showed an effect of suppressing elevation of blood insulin
level regardless of their molecular weights.
The potassium polyglutamates with a weight-average molecular weight of 12,000, in
particular, had an remarkably excellent effect in which the maximum insulin-level
elevation in blood was suppressed by 50% or more (to about 45% with respect to the
control).
[0076]
Table 5 Relative values of maximum insulin-level elevation in mouse (10-minute value
- initial value)
|
Maximum insulin-level elevation Average ± S.E. (pg/ml) |
Significant difference from control group |
Significant difference between sodium polyglutamate (weight-average molecular weight:
9,000)-administered group and potassium polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
127.4 ± 20.0 |
N.S. |
- |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
76.3 ± 10.1 |
P<0.05 |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight 6.000)-administered group |
88.6 ± 10.0 |
P<0.05 |
P<0.05 |
*) S.E.: Standard Error
*) N.S.: Not Significant |
[0077] It was found that when using the sodium polyglutamates with a weight-average molecular
weight of about 9,000, an effect of suppressing elevation of blood insulin level was
not recognized, whereas the potassium polyglutamates with a weight-average molecular
weight of 12,000 remarkably suppressed the maximum insulin-level elevation in blood.
It was further revealed that the potassium polyglutamates with a low molecular weight
having a weight-average molecular weight of 6,000 had an effect of suppressing elevation
of an insulin level similar to that obtained when using potassium polyglutamates having
a weight-average molecular weight of 12,000.
[0078] From the results of Test Examples 1 and 2, it was also found that when using potassium
polyglutamates, the molecular weight of the potassium polyglutamates became lower,
surprisingly the effect of suppressing elevation of blood insulin level became higher,
whereas when using sodium polyglutamates, the molecular weight of the sodium polyglutamates
became larger, the function of suppressing elevation of blood insulin level tended
to became higher. The viscosity of the salts of polyglutamic acids become lower as
decreasing the molecular weight of the salts of polyglutamic acids. Thus, when potassium
polyglutamates with low molecular weight can be used, handling in the production process
be easy. In addition, when the agent for suppressing elevation of blood insulin level
of the present invention is formed into an oral liquid agent, there is an advantage
to be able to obtain a liquid agent excellent in feeling of the throat, slimy feeling,
ease of swallowing and the like during ingestion, by using potassium polyglutamates
with low molecular weight.
[0079] As mentioned above, it is known that excessive elevation of blood insulin level leads
to insulin resistance, eventually causes obesity and diabetes mellitus. Thus, the
potassium polyglutamates can be preferably used for preventing or reducing obesity
and diabetes mellitus by suppressing insulin secretion after meal ingestion.
Measurement and evaluation of triglyceride level in blood:
[0080] The blood collected by the heparin-treated hematocrit capillary was stored under
ice-cooling until plasma separation, and centrifuged at 11,000 rpm for 5 minutes to
give plasma. Blood triglyceride level in the obtained plasma was measured by using
triglyceride E-test Wako (manufactured by Wako Pure Chemical Industries, Ltd., GPO·DAOS
method).
Based on the results of the measurement of the triglyceride levels in bloods up to
2 hours after the oral administration of the sample, the difference (Δ value) between
the maximum value and the initial value (at the time of the initial blood sampling)
of the blood triglyceride level was defined as the maximum triglyceride-level elevation,
and Table 6 (results of Test Example 1) and Table 7 (results of Test Example 2) show
the values when the value obtained from the control group defined as 100.
[0081] Further, the statistically-significant difference between the groups was also considered
based on the obtained values of the maximum triglyceride-level elevation, and is shown
in Table 6 and Table 7. Verifications were performed between the salts of polyglutamic
acids-administered groups and the control group, and between the sodium salts-administered
groups and similar molecular weight of the potassium salts-administered groups using
a multiple comparison test (Bonferroni/Dunn method). From the obtained result, significance
was judged with considering p<0.05 as a significant difference.
[0082]
Table 6 Relative values of maximum triglyceride-level elevation in mouse (maximum
value - initial value)
|
Maximum triglyceride-level elevation Average ± S.E. (mg/dl) |
Significant difference from control group |
Significant difference between sodium polyglutamate-administered group and potassium
polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamates (weight-average molecular weight: 9,000)-administered group |
-47.3± 30.3 |
P<0.05 |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight 12,000)-administered, group |
-144.4 ± 33.2 |
P<0.05 |
Sodium polyglutamate (weight-average molecular weight: 350,000)-administered group |
15.1 ± 16.6 |
P<0.05 |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 240,000)-administered group |
-59.5 ± 11.9 |
P<0.05 |
*) S.E.: Standard Error
*) N.S.: Not Significant |
[0083] From the results of Table 6, it was found that in all cases in which the salts of
polyglutamic acids were administered, the maximum triglyceride-level elevation could
be effectively suppressed or lowered.
In addition, when potassium polyglutamates were administered, further remarkable effect
of lowering blood triglyceride level was recognized. This can be understood from that
a lowering effect obtained by using the potassium polyglutamates with a weight-average
molecular weight of 12,000 exceeding suppressing effect of the maximum triglyceride-level
elevation was more excellent than those obtained by using sodium polyglutamates with
a weight-average molecular weight of 9,000.
[0084]
Table 7 Relative values of maximum triglyceride-level elevation (Maximum value - initial
value)
|
Maximum triglyceride-level elevation Average ± S.E. (mg/dl) |
Significant difference from control group |
Significant difference between sodium polyglutamate (weight-average molecular weight:
9,000)-administered group and potassium polyglutamate-administered group |
Control group |
100.0 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
-31.9 ± 22.1 |
N.S. |
- |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
-181.3 ± 40.6 |
P<0.05 |
P0.05 |
Potassium polyglutamate (weight-average molecular weight: 6,000)-administered group |
-144.5 ± 17.4 |
P<0.05 |
P<0.05 |
*) S.E. : Standard Error
*) N.S. : Not Significant |
[0085] From the results of Table 7, it was reconfirmed that when the potassium polyglutamates
were administered, a remarkably excellent effect of lowering blood triglyceride level
was recognized as compared to the case where the sodium polyglutamates were administered.
In addition, it was ascertained that the effect of lowering blood triglyceride level
exerted by the potassium polyglutamate with a weight-average molecular weight of 6,000
was the same level as that obtained by using potassium polyglutamates with a weight-average
molecular weight of 12,000.
[0086] As mentioned above, it is known that elevation of blood triglyceride level causes
hyperlipidemia and eventually arteriosclerosis. Thus, the above potassium polyglutamates
can be preferably used for preventing or reducing hyperlipidemia and arteriosclerosis
by effectively lowering blood triglyceride level after meal ingestion.
Measurement and evaluation of blood glucose level:
[0087] Using blood collected by a heparin-treated hematocrit capillary, blood glucose levels
were immediately measured using a simple blood sugar monitoring meter ACCU-CHEK (trade
name, manufactured by Roche Diagnostics K.K.).
The blood glucose level up to 2 hours after the oral administration of the sample
was measured. As a result, it was found that the blood glucose level became the maximum
at 10 minutes after the administration. Therefore, the difference (Δ value) between
the maximum value (at 10 minutes after the administration) and the initial value (at
the time of the initial blood sampling) of the blood glucose level was defined as
the maximum blood glucose-level elevation, and Table 8 show the values when the value
obtained from the control group defined as 100.
[0088] The statistically-significant difference between the groups was also studied based
on the obtained values of the maximum blood glucose-level elevation, and is shown
in Table 8. When significance (p<0.05) was recognized by an analysis of variance,
verifications were performed between the salts of polyglutamic acids-administered
groups and the control group, and between the sodium salts-administered groups and
similar molecular weight of the potassium salts-administered groups using a multiple
comparison test (Bonferroni/Dunn method). From the obtained result, significance was
judged with considering p<0.05 as a significant difference.
[0089]
Table 8 Relative values of maximum blood glucose-level elevation in mouse (10-minute
value - initial value)
|
Maximum blood glucose-level elevation Average ± S.E. (mg/dL) |
Significant difference from control group |
Significant difference between sodium polyglutamate-administered group and potassium
polyglutamate-administered group |
Control group |
100 |
- |
- |
Sodium polyglutamate (weight-average molecular weight: 9,000)-administered group |
99 ± 4 |
N.S. |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 12,000)-administered group |
86 ± 3 |
P<0.05 |
Sodium polyglutamate (weight-average molecular weight: 350,000)-administered group |
104 ± 3 |
N.S. |
P<0.05 |
Potassium polyglutamate (weight-average molecular weight: 240.000)-administered group |
88 ± 4 |
P<0.05 |
*) S.E.: Standard Error
*) N.S.: Not Significant |
[0090] From the results of Table 8, it was found that the sodium polyglutamates did not
have the function of suppressing elevation of blood glucose level regardless of their
molecular weights. In contrast, in the groups to which the potassium polyglutamates
were administered, the elevation of blood glucose level was significantly suppressed
as compared to both the control group and the sodium salts-administered groups. From
the above results, it was found that by employing potassium polyglutamates as active
ingredients of the agent for suppressing elevation of blood glucose level, a remarkable
effect of suppressing elevation of blood glucose level, which could not be obtained
using other polyglutamic acid salts, could be obtained.
[0091] As mentioned above, it is known that rapid elevation of blood glucose level after
meal ingestion leads to insulin resistance, eventually causes diabetes mellitus, obesity
and arteriosclerosis. Thus, the above potassium polyglutamates can be preferably used
for preventing or reducing diabetes mellitus, obesity and arteriosclerosis by effectively
suppressing rapid elevation of blood glucose level.
[0092] Having described our invention as related to the present embodiments, it is our intention
that the invention not be limited by any of the details of the description, unless
otherwise specified, but rather be construed broadly within its spirit and scope as
set out in the accompanying claims.
[0093] This application claims priority on Patent Application No.
2011-004313, Patent Application No.
2011-004314, Patent Application No.
2011-004315 and Patent Application No.
2011-004316 filed in Japan on January 12, 2011, which are entirely herein incorporated by reference.